Creatinine is a metabolic waste product formed by non-enzymatic dehydration of creatine which is produced from creatinephosphoric acid, one of the sources of muscular contractile energy. Creatinine is not utilized in vivo and is excreted as an end metabolite in urine. Creatinine also exists in blood in a normal concentration of about 0.7-1.5 mg/100 ml of serum. The determination of the amount of creatinine in blood and urine is, therefore, very useful for diagnostic purposes in the determination of kidney diseases such as acute nephritis and chronic nephritis and of disorders such as urethrophraxis, mercurialism, nephrosis, etc.
Heretofore, the quantitative determination of creatinine has been carried out according to colorimetric determination utilizing Jaffe's reaction which comprises a color reaction with alkaline picrate. While colorimetric determination of creatinine using alkaline picrate is practical in that the operation is simple and stable, this method involves certain defects. That is, because of poor sensitivity, the use of an increased amount of serum is necessary; the reaction is not specific; and the reaction is subject to the influence of substances in the blood such as an active methylene compound, proteins, antibiotics, etc. In order to overcome these defects, it is necessary to remove the substances in the sample or to extract the creatinine. However, such operations complicate the procedure and, therefore, are disadvantageous for automatic operation.
It has now been found that the amount of creatinine in a sample can be determined more simply, more correctly and more speedily as compared to the heretofore known method, by decomposing creatinine using an enzyme which catalyzes a reaction wherein creatinine is hydrolyzed into N-methyl-hydantoin an ammonia and then measuring the amount of the formed N-methyl-hydantoin or ammonia.
The catalytic enzyme employed in the process of the present invention is creatinine desimidase.
Creatinine desimidase (EC 3.5.4.21) was reported by J. Szulmajster (J. Bacteriology 75, 633, 1958 and Biochimica Et Biophysica Acta 30, 1954, 1958) in the microbial cells of Clostridium paraputrificum in 1958. The enzyme participates in the decomposition of creatinine and catalyzes a reaction to hydrolyze creatinine to form N-methyl-hydantoin and ammonia.
However, microorganisms of the genus Clostridium are anaerobic and when they are used for the production of the enzyme, a long period of time is required for the fermentation, microbial growth is poor and the yield of the enzyme is poor. Therefore, it has not been possible to carry out the production of creatinine desimidase on an industrial scale.
Other prior investigators have reported findings of strains of aerobic organisms belonging to the genus Pseudomonas which could decompose creatinine. However, none of these prior investigators have reported a microorganism which produces an enzyme capable of catalyzing an hydrolysis reaction wherein creatinine is decomposed to form N-methyl-hydantoin and ammonia. Moreover, such prior investigators were unable, according to the published reports, to isolate a particular enzyme capable of catalyzing the above-mentioned reaction.
It has now been found that microorganisms of the genera Brevibacterium, Corynebacterium, Pseudomonas and Arthrobacter, when cultured in a nutrient medium, produce remarkable amounts of creatinine desimidase in the culture liquor and/or within the microbial cells.
The creatinine desimidase obtained according to the present invention may be readily used for the quantitative determination of the amount of creatinine in a sample in an automatic operation not heretofore attainable. More specifically, the amount of creatinine in a sample can be simply and quickly determined by measuring the amount of N-methyl-hydantoin or ammonia formed through the catalytic action of the enzyme.